trans

TRANSPORT SYSTEMS 205
forms of the parasite. Unlike Leishmania and
T. cruzi, T. brucei persist in an extracellular
form throughout their entire life cycle, and the
purine and pyrimidine environment that they
encounter is influenced by plasma concentrations.
Two types of high-affinity adenosine transport
activities have been identified in T. brucei
and other species of Trypanosoma: P1, which
transports adenosine, inosine and guanosine,
and P2, which exhibits a restricted specificity
for adenine and adenosine. The P2 transporter
is distinguished by its ability to transport
two classes of anti-trypanosomal drugs
with high affinity: the diamidines and the
melaminophenyl arsenicals. Trypanosomes
resistant to these classes of drugs either lack
or have significantly impaired P2 transport
activity. Recently the P2 gene, or TbAT1, was
cloned by functional complementation using
yeast purine auxotrophs that are naturally
deficient in adenosine transport. TbAT1 is a
single-copy gene and encodes an ENT-type
protein of 436 amino acids that shares limited
sequence identity with LdNT1 and LdNT2 and
the human ENTs 1 and 2. When expressed
in yeast, TbAT1 confers sensitivity to the
melaminophenyl arsenical drug, melarsen
oxide, although TbAT1-mediated adenosine
transport was not susceptible to the diamidine
drugs pentamidine and berenil. Isolation
of the TbAT1 allele from a melaminophenyl
arsenical-resistant isogenic strain revealed
nine point mutations within the mutant
TbAT1 allele, six of which result in amino
acid substitutions. Subsequent analysis of 65
melaminophenyl arsenical-resistant field isolates
identified eight of the same nine mutations
with an additional deletion of an entire
codon.
A gene encoding P1 activity has also been
cloned from T. brucei. This gene, TbNT2,
shares considerable homology to TbAT1 (58%
identity at the amino acid level), but upon
expression in Xenopus oocytes displays a
P1-type activity that transports adenosine,
inosine, and guanosine with high affinity.
Southern analyses and searches of the T. brucei
genomic database have revealed a surprising
molecular complexity at the TbNT2 locus.
TbNT2 is part of a multigene cluster on chromosome
11, comprising some six members
that all share 81–96% sequence identity at the
amino acid level.
Nucleobase transport appears to be mediated
by several stage-specifically expressed
transport activities with overlapping ligand
specificity. In bloodstream T. brucei, two activities
have been delineated, H2 and H3. H2 is
a broad-specificity purine and pyrimidine
nucleobase transporter, which also recognizes
guanosine and possibly inosine. H3 is a highaffinity
transporter with a substrate specificity
restricted to the purine nucleobases. In procyclic
T. brucei, two nucleobase transport
activities have been demarcated, H1 and U1.
H1 is strikingly similar to the H3 activity
described in the bloodstream stage of the parasite.
Indeed, H1 and H3 can only be differentiated
by their respective affinities for
xanthine and the pyrazolopyrimidine, allopurinol.
U1 is a high-affinity, uracil-specific
transporter, which does not appear to efficiently
recognize any other pyrimidine nucleobase
or nucleoside, although it may transport
uridine with low affinity.
Although electrophysiological measurements
have not been performed, it appears
that all of the T. brucei nucleobase and nucleoside
transporters couple transport to the
translocation of a proton. Destruction of the
proton gradient across the parasite plasma
membrane with protonophores or metabolic
inhibitors results in a loss of transport
capability for all of the transporters described
above.
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA